The 2nd generation sequencing technologies of Illumina Solexa, AB SOLiD and Roche 454 achieve their high throughput and relatively low costs per sequenced base by operating in a massively parallel fashion. They create many random fragments of the total input DNA, amplify each fragment separately on a bead or on an array, read out the sequences of those in a single flow cell, and are able to detect mutations only by matching all individual reads to a reference genome sequence (Margulies M. et al. (2005) Nature 437, 376-380). This way of operation has the drawback that many irrelevant parts of the genome are sequenced as well. This will especially be true for clinical sequencing, were usually sets of only tens to hundreds of genes are relevant for a diagnosis. Therefore, different technologies are currently in use to select relevant parts of the genomic DNA prior to introducing the sample to the sequencer. Most commonly, the DNA is hybridized to capture probes on a microarray that specifically bind the relevant parts. In a second step, the unbound, irrelevant parts are washed away, and subsequently the bound fragments are eluted and prepared to be loaded into the sequencer (Okou D. et al. (2007) Nat Methods 4, 907-909). Typically, target fractions of 0.1%-1% of a genome can be enriched to 60-80% in the preselected sample. In this workflow, the processing of information is not optimal. During preselection on the array, information is available about the genomic location of the DNA fragments, because they are specifically bound to probes that are spatially separated and can be identified. Nevertheless, the elution of the array is a one-chamber process, so that all selected fragments become mixed again. Consequently, after sequencing all reads have to be matched individually to a complete reference sequence, requiring a large computational effort. If one would be able to keep the genomic information from the preselection array into the sequencing reaction, this would significantly reduce computation requirements. The simplest way to retain this information is to do preselection and sequencing on the same spot on the same surface. Such methods are described in US application US2009/0117573. Since current sequencing technologies need amplification of every individual DNA fragment to obtain sufficient signal during sequencing, this amplification take places on the same spot. Based on the principle of bridge amplification technology, which is a surface-attached amplification that is for example used in Illumina's sequencing technology, a method is described to solve this. This can be performed on any solid surface (slides, carriers etc.) as long as individually amplified colonies can be discerned.
In these methods different hybridization, extension and ligation steps are used before sequencing can take place. More efficient and faster methods are still required.